Enhanced Biodegradation of Pesticides in the Environment - American

Chapter 9

The Role of Fungi and Bacteria in the Enhanced Degradation of the Fungicide Carbendazim and the Herbicide Diphenamid 1

2

1

2

N. Aharonson , J. Katan , E. Avidov , and O. Yarden 1

Department of Chemistry of Pesticides and Natural Products, Agricultural Research Organization, The Volcani Center, Bet Dagan, 50-250, Israel Department of Plant Pathology and Microbiology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, 76-100, Israel

Downloaded by MONASH UNIV on February 28, 2016 | http://pubs.acs.org Publication Date: May 3, 1990 | doi: 10.1021/bk-1990-0426.ch009

2

Single application of carbendazim (MBC) or repeated applications of diphenamid conditioned the soil for enhanced degradation. Low i n i t i a l doses of the pesticide or mixing a small volume of carbendazim-history soil with untreated soil sufficed to condition the entire soil volume for enhanced degradation. Soil disinfestation or treatments with fungicides such as triphenyltin acetate or TMTD were generally effective in decreasing enhanced degradation of the two pesticides. Fungi capable of degrading MBC or diphenamid were isolated, enumerated and identified from soils with and without enhanced degradation. There were no differences in the degradation between fungi isolated from previously treated or nontreated soils. Differences were found only between mixed bacterial cultures. These results, together with data about cross enhancement between some metabolites and their respective parent compounds, and the similarity in the metabolism between soil and bacterial cultures suggests that for these pesticides bacteria are the major component responsible for enhanced degradation, though fungi degraders are also present in the s o i l . The work suggests possible enzymatic reactions related to enhanced degradation.

Enhanced recent

degradation

years

nematicides to

for

enhanced

articles soil

classes

of

number o f

.

was

well

degradation

the and

same o r the

a

has

insecticides, of

and which

Enhanced degradation of

pesticides

The d i v e r s i t y

chemicals

degradation,

application

Enhanced

soil-applied

and h e r b i c i d e s .

different

with

of

a growing

pesticides, were

documented is

structurally

found

in

usually

accompanying

been

reported belonging

associated

several

review

linked with related

failure

in

fungicides,

of

repeated

pesticide.

pest

control

0097-6156/90/0426-0113$06.00/0 © 1990 American Chemical Society

In Enhanced Biodegradation of Pesticides in the Environment; Racke, Kenneth D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


114

ENHANCED BIODEGRADATION OF PESTICIDES IN T H E ENVIRONMENT

have c r e a t e d an i n c r e a s i n g l y s e r i o u s a g r i c u l t u r a l problem f o r f a r m e r s and have r a i s e d some fundamental q u e s t i o n s w i t h r e g a r d t o s h i f t s i n m i c r o b i a l populations i n s o i l (5-8). I t i s g e n e r a l l y a c c e p t e d t h a t m i c r o b i a l metabolism as e x p r e s s e d by p o p u l a t i o n s h i f t s and/or a c t i v i t y o f d e g r a d e r s a r e the major f a c t o r s c o n t r i b u t i n g t o enhanced d e g r a d a t i o n and c o n v e r s i o n o f p e s t i c i d e s t o b i o l o g i c a l l y n o n - a c t i v e compounds. T h i s has been d e m o n s t r a t e d i n e x p e r i m e n t s i n which the p r o c e s s o f enhanced d e g r a d a t i o n was s t o p p e d o r d e l a y e d by s o i l s t e r i l i z a t i o n o r by a n t i m i c r o b i a l t r e a t m e n t s (9,10). The m i c r o b i a l p o p u l a t i o n e x i s t s i n a dynamic e q u i l i b r i u m t h a t can be a l t e r e d by m o d i f y i n g e n v i r o n m e n t a l c o n d i t i o n s (e.g. a d d i t i o n o f p e s t i c i d e s o r o t h e r b i o l o g i c a l l y a c t i v e s u b s t a n c e s ) . M i c r o o r g a n i s m s may respond t o p e s t i c i d e s as s u b s t r a t e s and t h e r e b y d e r i v e energy o r u t i l i z a b l e n u t r i e n t s f o r metabolism. The p e s t i c i d e may a l s o undergo d e g r a d a t i o n by a n a l o g - i n d u c e d o r c o n s t i t u t i v e cometabolism, whereby the p e s t i c i d e i t s e l f does n o t s e r v e as an energy s o u r c e ( 1 , 3 ) . The e x a c t mechanisms f o r m i c r o b i a l a d a p t a t i o n t o the p e s t i c i d e m o l e c u l e i n s o i l s t h a t d e v e l o p enhanced d e g r a d a t i o n c a p a c i t y a r e n o t c o m p l e t e l y u n d e r s t o o d . These p r o c e s s e s c o u l d be viewed from the e c o l o g i c a l and p o p u l a t i o n a s p e c t s , from t h e i r b i o c h e m i c a l and e n z y m a t i c r e a c t i o n s , o r from the g e n e t i c a s p e c t s , i n which extrachromosomal elements may be i n v o l v e d as p a r t o f the p r o c e s s . T h i s summary p r e s e n t s d a t a on two p e s t i c i d e s w i t h . d i f f e r e n t m o l e c u l a r s t r u c t u r e s and b i o l o g i c a l a c t i v i t i e s , w i t h the emphasis on t h e m i c r o o r g a n i s m s a s s o c i a t e d w i t h the development o f enhanced d e g r a d a t i o n and the p o s s i b l e enzymatic r e a c t i o n s t h a t a r e taken p l a c e i n these processes. Enhanced

Degradation

P r e v i o u s a p p l i c a t i o n o f the same p e s t i c i d e o r o f a s t r u c t u r a l l y r e l a t e d compound i s the major f a c t o r i n i n d u c i n g enhanced d e g r a d a t i o n . I n c e r t a i n c a s e s enhanced d e g r a d a t i o n might d e v e l o p i n t h e s o i l f o l l o w i n g one p r e t r e a t m e n t . The f u n g i c i d e carbendazim (methyl b e n z i m i d a z o l - 2 - y l c a r b a m a t e , MBC) r e p r e s e n t s such a group o f compounds. A f t e r one t r e a t m e n t t h e h a l f - l i f e o f MBC was s h o r t e n e d from 17 days t o 3-^4 days (11 and F i g u r e 1 ) . F o r some p e s t i c i d e s even t h e s m a l l amount t h a t r e a c h e s the s o i l t h r o u g h f o l i a r s p r a y a p p l i c a t i o n s i s s u f f i c i e n t t o i n d u c e enhanced d e g r a d a t i o n , as has been shown by Walker e t a l . w i t h the f u n g i c i d e i p r o d i o n e (12) and by Yarden e t a l . w i t h the f u n g i c i d e MBC ( 1 1 ) . On the o t h e r hand, the maximal r a t e o f enhanced d e g r a d a t i o n was o b t a i n e d w i t h compounds such as the h e r b i c i d e diphenamid (Ν,Ν-dimethyl 2 , 2 - d i p h e n y l a c e t a m i d e ) , o n l y a f t e r s e v e r a l r e p e a t e d t r e a t m e n t s . Four s o i l t r e a t m e n t s w i t h diphenamid were needed b e f o r e i t s h a l f - l i f e was d e c r e a s e d from 50-60 days t o 3-k days ( 9 ) . I t i s not s u r p r i s i n g t h a t enhanced d e g r a d a t i o n has d e v e l o p e d t o v a r y i n g degrees i n d i f f e r e n t s o i l s and under d i f f e r e n t a g r i c u l t u r a l regimes. T h i s c o u l d be the r e s u l t o f t h e i n t r i n s i c c h e m i c a l p r o p e r t i e s o f the p e s t i c i d e , b u t c o u l d a l s o d e r i v e from the f a c t t h a t , among o t h e r p r o c e s s e s , c e r t a i n s o i l s a r e " s u p p r e s s i v e " w h i l e o t h e r s a r e " c o n d u c i v e " t o enhanced d e g r a d a t i o n , s i m i l a r t o the w e l l e s t a b l i s h e d phenomenon w i t h s o i l b o r n e pathogens ( 3 ) . in

A l t h o u g h MBC and diphenamid d i f f e r c o n s i d e r a b l y from each o t h e r the ease w i t h which they i n d u c e enhanced d e g r a d a t i o n , t h e i r


AHARONSONETAL.


9.

0

115

Roles of Fungi & Bacteria

2

4

6

8

10

12

DAYS OF INCUBATION Figure

1.

of

percent

1-9

capacity mixed from

D i p h e n a m i d a n d MBC d e g r a d a t i o n (P)

with Ref.

of

with

soil

that

has

non-history

autoclaved

soil

acquired

soil

(NP)

and s t e r i l e

in

mixtures

enhanced and,

sand.

for

composed

degradation MBC s o i l ,

MBC d a t a

was

also adapted

11.


116

ENHANCED BIODEGRADATION O F PESTICIDES IN T H E ENVIRONMENT

behavior was of

in

found a

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pesticide,

thiocarbamate), diphenamid. suggest also

a

for An

formed

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the

absence

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a l . ,

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Such

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under

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the

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that

Similar

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conditions, of

the

the

nutrients

spread

to

sharp

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of

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the

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(Figure

(12). The i m p l i c a t i o n s

for

the

treated

pronounced. for

in

mixing

diphenamid

explained

abundance

developed

treated

mixtures

autoclaved

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for

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has

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soilborne

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1%

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(S-ethyl

non-problem

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by which

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2%

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be

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with

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of

The major n o t i c e a b l e

1).


the

with

to

similarities,

mechanism

degradation,

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found

these

by m i x i n g

soils

herbicide

generalization

additional

induced

the

classes

is

(i.e.,

respects

also

of

certain

degradation past

was

Some

other

enhanced

soils

many o t h e r

the these

this

enhanced

phenomenon

of

soil

pathogens.

Disinfestation

and A n t i m i c r o b i a l

Agents

on

Enhanced

Degradation Soil

Disinfestation.

solarization nontreated

soils

degradation nontreated and

(Figure

(Figure in

non-disinfested

enhanced

the

of

rate

in

the

was

Soil

even

control

slower

in

with

treatment microbial

in

autoclaving

disinfestation

showing that

can

enhanced be

degradation under

these

greenhouse

disinfestaion

or

for

field and

the

constitutes

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Moreover, purpose

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This

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that

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a general

broad-spectrum

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incomplete

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soil

Therefore,

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soil, data

as

the

degradation

microorganisms well

in as

the in

p o i n t .towards

controlling is

that

degradation

soil means

enhanced

especially

cropping conditions,

regular

soils

The d e g r a d a t i o n

suppressing

o f MBC

the

diphenamid

a drastic, be

in enhanced

degradation

gamma i r r a d i a t i o n .

conditions.

intensive

MBC o r

nontreated

and can

important

pesticides

degradation.

developed under

the

soil

previously

some c a s e s

regarded

activity,

of

of

of

slower

degradation

in

results

by

demonstrates

or

had developed

disinfestation, and

be

which

in

either

the

sterilization clearly

that

pesticides.

soil,

effectiveness degradation

bromide

The d i s i n f e s t a t i o n

after

can

the

for

both

than

disinfestation

antimicrobial reduction

of

soil,

soils

resulted

soils.

degradation

methyl

disinfestation

soils

degradation

degradation

nontreated

former

2) a n d i n

comparison

with

MBC a n d d i p h e n a m i d d e g r a d a t i o n

3). F i e l d

control

acquired

fumigation

reduced

(non-problem)

diphenamid

enhanced

Soil

strongly

relevant

where

soil

practice.



9.

117


AHARONSONETAL.

DAYS AFTER TREATMENT Figure

2.

Effect

solarization, acetate soil. Data

( F A ) , on

The from

of

soil

autoclaving the

fungicides

disinfestation and

treatment

degradation TMTD

Phytopathology.

of 75,

methyl

TMTD a n d

MBC t h a t

and FA were 1985,

with with

added

was at

bromide

added

20 U g / g

(MB),

fentin at

10

Pg/g

soil.

763.


118


E N H A N C E D BIODEGRADATION O F PESTICIDES IN T H E ENVIRONMENT

0

10

0

5

20

10 DAYS

Figure the

3. E f f e c t

degradation

enhanced

of soil

30

15

disinfestation

degradation

capacity

( N P ) . MB=methyl b r o m i d e ,

from

Ref.9

50

20

AFTER TREATMENT a n d some

o f d i p h e n a m i d a n d MBC i n s o i l s

soil

40

fungicides

that

( Ρ ) , as compared w i t h FA=fentin acetate.

have a

Data

on

acquired

nontreated adapted

and R e f . l l .


9.

AHARONSONETAL.


119

S o i l d i s i n f e s t a t i o n w i t h methyl bromide, vapam, o r by s o l a r i z a t i o n c o n t r o l l e d d e g r a d a t i o n o f c e r t a i n p e s t i c i d e s such as EPTC t o v a r i o u s degrees ( 1 3 ) . A p p a r e n t l y some o f t h e d e g r a d e r s were n o t a f f e c t e d by t h e s e b i o c i d e s , a f a c t t h a t was r e f l e c t e d by p a r t i a l r e d u c t i o n i n d e g r a d a t i o n o f EPTC, when compared w i t h t h e s t r o n g e r inhibitory effect i n sterile soil.


E f f e c t o f Some F u n g i c i d e s and Other A n t i m i c r o b i a l Agents. I t i s e x p e c t e d t h a t t h e u s e o f more s p e c i f i c b i o c i d e s , which have a n a r r o w e r spectrum o f a n t i m i c r o b i a l a c t i v i t y as compared w i t h d i s i n f e s t a t i o n , w i l l a l s o r e s u l t i n s l o w i n g down d e g r a d a t i o n o f c e r t a i n p e s t i c i d e s i n r e g u l a r s o i l s o r i n s o i l s showing enhanced d e g r a d a t i o n . However, t h e r e d u c t i o n i n r a t e s o f d e g r a d a t i o n would depend s t r o n g l y on t h e b i o c i d e i n q u e s t i o n and on i t s c o n c e n t r a t i o n

(14). C e r t a i n b i o c i d e s e x h i b i t a r e l a t i v e l y narrow range o f a c t i v i t y and t h e r e f o r e might be u s e f u l f o r i d e n t i f i c a t i o n o f t h e d e g r a d e r s a s s o c i a t e d w i t h enhanced d e g r a d a t i o n . C l a s s i c a l p a r a m e t e r s , such as t o t a l m i c r o b i a l biomass, e n z y m a t i c a c t i v i t y , m i c r o b i a l r e s p i r a t i o n and e n u m e r a t i o n , a r e v e r y rough measurements o f m i c r o b i a l a c t i v i t y . Thus, when d e a l i n g w i t h t h e d e g r a d a t i o n o f p e s t i c i d e s , which a r e a p p l i e d t o t h e s o i l i n v e r y s m a l l amounts, such p a r a m e t e r s might n o t be s e n s i t i v e enough t o c o r r e l a t e w i t h r a t e s o f d e g r a d a t i o n ( 1 5 ) . A p p l i c a t i o n o f c e r t a i n b i o c i d e s , such as c y c l o h e x i m i d e , PCNB, o x y t e t r a c y c l i n e , c a p t a n , c h l o r a m p h e n i c o l o r TMTD, which demonstrate a c e r t a i n degree o f s p e c i f i c i t y , may s e r v e as an a d d i t i o n a l u s e f u l t o o l f o r the i d e n t i f i c a t i o n o f m i c r o b i a l degraders i n s o i l s with enhanced d e g r a d a t i o n (16). However, i t s h o u l d be p o i n t e d o u t t h a t e m p l o y i n g b i o c i d e s f o r i d e n t i f i c a t i o n s h o u l d be c o n s i d e r e d w i t h c a u t i o n , s i n c e t h e s e b i o c i d e s a r e o f t e n n o t v e r y s p e c i f i c and may a f f e c t a l a r g e r group o f m i c r o o r g a n i s m s . The c o n c e n t r a t i o n o f t h e b i o c i d e i s v e r y c r i t i c a l i n d e t e r m i n i n g i t s degree o f s p e c i f i c i t y . The r e s u l t s f o r t h r e e f u n g i c i d e s - b i o c i d e s i n c o n t r o l l i n g d e g r a d a t i o n were as f o l l o w s : The f u n g i c i d e s t e t r a m e t h y l t h i u r a m d i s u l f i d e (TMTD) and t r i p h e n y l t i n a c e t a t e ( f e n t i n a c e t a t e ) were v e r y e f f e c t i v e i n delaying degradation o f several s o i l - a p p l i e d p e s t i c i d e s ( F i g u r e 3 ) . F e n t i n a c e t a t e was found as e f f e c t i v e as t h e b r o a d - s p e c t r u m b i o c i d e methyl bromide i n i n h i b i t i n g d e g r a d a t i o n o f diphenamid i n a s o i l w i t h enhanced d e g r a d a t i o n c a p a c i t y , whereas t h e f u n g i c i d e s TMTD and TBZ were l e s s a c t i v e . These f i n d i n g s a l s o d e m o n s t r a t e t h e p o t e n t i a l o f s p e c i f i c c h e m i c a l s t o s u p p r e s s enhanced d e g r a d a t i o n . The e f f e c t i v e n e s s o f v a r i o u s f u n g i c i d e s i n s u p p r e s s i n g enhanced d e g r a d a t i o n may s u g g e s t t h a t f u n g i a r e i n v o l v e d i n t h e enhanced d e g r a d a t i o n o f MBC o r diphenamid. A g a i n , t h i s might be q u e s t i o n a b l e s i n c e t h e e f f e c t i v e n e s s o f many f u n g i c i d e s extends beyond f u n g i , e s p e c i a l l y when t h e f u n g i c i d e s a r e a p p l i e d a t h i g h c o n c e n t r a t i o n s . F o r example, t h e f u n g i c i d e PCNB i s a l s o v e r y t o x i c to actinomycetes (17). Microbial

Studies

Enhanced d e g r a d a t i o n i s t h e r e s u l t o f m i c r o b i a l p r o c e s s e s i n which an e n r i c h m e n t i n t h e p o p u l a t i o n o r a c t i v i t y o f m i c r o o r g a n i s m s c a p a b l e o f d e g r a d i n g t h e p e s t i c i d e , o r i n d u c t i o n o f enzymatic


120



r e a c t i o n s a l r e a d y p r e s e n t i n the s o i l m i c r o f l o r a , a r e e x p e c t e d . G e n e r a l c o u n t s o f the t o t a l p o p u l a t i o n s o f b a c t e r i a , f u n g i and actinomycetes d i d not r e v e a l s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s r e g a r d i n g diphenamid and MBC-history and n o n h i s t o r y s o i l s , as w i l l be d e s c r i b e d . T h i s i s n o t s u r p r i s i n g , s i n c e the d e g r a d e r s c o n s t i t u t e o n l y a s m a l l p r o p o r t i o n o f the t o t a l s o i l m i c r o f l o r a . T h e r e f o r e , even i f a change o c c u r s i n segments o f the p o p u l a t i o n , i t might n o t be d e t e c t e d through a g e n e r a l count. F u n g a l D e g r a d e r s . I n o r d e r t o a s s e s s the d e g r a d a t i v e c a p a b i l i t i e s o f f u n g i , i n d i v i d u a l c o l o n i e s from each t e s t e d s o i l were randomly t r a n s f e r r e d t o p o t a t o d e x t r o s e agar amended w i t h 10 ug/g MBC and a s s e s s e d f o r t h e i r c a p a b i l i t y t o degrade the f u n g i c i d e ' ( 1 9 ) . I r r e s p e c t i v e o f the o r i g i n o f the i s o l a t e s , n e a r l y 80% were i d e n t i f i e d as A l t e r n a r i a a l t e r n a t a , B i p o l a r i s o r U l o c l a d i u m sp. An MBC-degrading fungus i s o l a t e d from MBC-history s o i l was i d e n t i f i e d as Acremonium f a l c i f o r m e . The r e s u l t s showed t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the number o f d e g r a d e r s o r i n the r a t e o f d e g r a d a t i o n by t h e f u n g i p o p u l a t i o n s t h a t were i s o l a t e d from a s o i l w i t h o r w i t h o u t enhanced d e g r a d a t i o n . A s i m i l a r experiment was c a r r i e d out w i t h f u n g i c a p a b l e o f d e g r a d i n g diphenamid, i s o l a t e d from diphenamid h i s t o r y o r n o n h i s t o r y s o i l s ( 1 8 ) . The c a p a c i t y o f the v a r i o u s f u n g i t o degrade diphenamid v a r i e d , and the d i s t r i b u t i o n o f the d e g r a d e r s was about the same i n b o t h s o i l s . Most o f the f u n g i t h a t were i s o l a t e d from the two s o i l s were c a p a b l e o f d e g r a d i n g diphenamid t o a c e r t a i n e x t e n t . The most e f f i c i e n t f u n g a l d e g r a d e r s were i d e n t i f i e d as Fusarium, A s p e r g i l l u s and Pénicillium spp. From t h i s s t u d y i t was c o n c l u d e d t h a t f u n g i p r o b a b l y were n o t the major organisms r e s p o n s i b l e f o r the enhanced d e g r a d a t i o n o f diphenamid and MBC, even though many o f them might be i m p o r t a n t i n "normal" d e g r a d a t i o n o f t h e s e p e s t i c i d e s i n r e g u l a r s o i l s b e f o r e t h e l a t t e r d e v e l o p e d enhanced d e g r a d a t i o n . However, i n such s t u d i e s i t i s s t i l l an open q u e s t i o n whether the b e h a v i o r o f t h e s e f u n g i i n c u l t u r e r e f l e c t s t h e i r c a p a c i t y t o degrade the p e s t i c i d e i n the soil. B a c t e r i a l Degraders. The main d i f f e r e n c e between s o i l s w i t h and w i t h o u t enhanced d e g r a d a t i o n , w i t h r e g a r d t o m i c r o b i a l d e g r a d e r s , was o b s e r v e d i n mixed b a c t e r i a l c u l t u r e s o r i g i n a t i n g from the two s o i l s . The d e g r a d a t i o n o f MBC (19), diphenamid (18) and EPTC (20) was found t o be much f a s t e r i n mixed b a c t e r i a l c u l t u r e s o r i g i n a t i n g from s o i l s t h a t d e v e l o p e d enhanced d e g r a d a t i o n c a p a c i t y than from n o n h i s t o r y s o i l s ( F i g u r e 4 ) . The r o l e o f t h e s e organisms i n enhanced d e g r a d a t i o n was demonstratead by i n o c u l a t i n g them i n t o n o n h i s t o r y s o i l s , which r e s u l t e d i n an enhancement o f d e g r a d a t i o n i n the l a t t e r s o i l s . F u n g i were not p r e s e n t i n t h e s e b a c t e r i a l c u l t u r e s . The h i g h pH ( 7 . 5 ) , c o n t i n u o u s s h a k i n g o f the medium, and the e a r l y p r e d o m i n a t i o n i n the medium by the b a c t e r i a , p r o v i d e d an u n f a v o r a b l e environment f o r f u n g i growth. The p o s s i b i l i t y t h a t a c t i n o m y c e t e s might be i n v o l v e d i n the enhanced d e g r a d a t i o n o f diphenamid was examined by a p p l y i n g t o the c u l t u r e PCNB, which i s i n h i b i t o r y t o a c t i n o m y c e t e s . The medium used f o r e s t i m a t i n g the number o f s o i l b a c t e r i a a l s o p e r m i t s the


AHARONSONETAL.



9.


121

122


development o f a c t i n o m y c e t e c o l o n i e s . PCNB, as d e s c r i b e d by F a r l e y and Lockwood (17), was found t o i n h i b i t growth o f a c t i n o m y c e t e s i n c u l t u r e media whereas b a c t e r i a were u n a f f e c t e d . A c t i n o m y c e t e s numbers were r e d u c e d 90% by 10 yg/ml PCNB, and 99% by 25-200 Ug/ml. Numbers o f b a c t e r i a were not reduced i n s o i l e x t r a c t s , even a t PCNB c o n c e n t r a t i o n s as h i g h as 200 yg/ml.


In t h i s experiment, a t a c o n c e n t r a t i o n o f 10 Ug/ml o f PCNB i n the mixed b a c t e r i a l c u l t u r e , 99% o f the a c t i n o m y c e t e s p r o p a g u l e s i n t h e c u l t u r e were e l i m i n a t e d w i t h o u t any e f f e c t on diphenamid d e g r a d a t i o n . On the o t h e r hand, the a d d i t i o n o f the b a c t e r i c i d e c h l o r a m p h e n i c o l t o the b a c t e r i a l c u l t u r e s o b t a i n e d from diphenamid h i s t o r y s o i l s s t r o n g l y i n h i b i t e d diphenamid d e g r a d a t i o n (Table I ) . The f a c t t h a t some f u n g i c i d e s such as TMTD and f e n t i n a c e t a t e were v e r y e f f e c t i v e i n c u r b i n g enhanced d e g r a d a t i o n o f s e v e r a l p e s t i c i d e s , l e d us t o c o n s i d e r the p o s s i b i l i t y t h a t f u n g i c o u l d be i n v o l v e d i n the enhanced d e g r a d a t i o n . However, i t has been shown t h a t t h e s e f u n g i c i d e s , a t the t e s t e d c o n c e n t r a t i o n s , were a l s o v e r y e f f e c t i v e i n c o n t r o l l i n g the b a c t e r i a l d e g r a d e r s i n the mixed b a c t e r i a l c u l t u r e s . The a d d i t i o n o f 10 Pg/ml o f each o f the f u n g i c i d e s t o the b a c t e r i a l c u l t u r e slowed down the d e g r a d a t i o n t o the same r a t e as t h a t o f c u l t u r e s from n o n - h i s t o r y s o i l s , whereas the a n t i f u n g a l agent c y c l o h e x i m i d e (50 pg/ml) d i d n o t a f f e c t the degradation (Table I ) . T a b l e I . D e g r a d a t i o n o f Diphenamid (10 pg/ml) i n Mixed B a c t e r i a l C u l t u r e , D e r i v e d from S o i l w i t h Enhanced D e g r a d a t i o n C a p a c i t y , and which was Amended w i t h Thiram (TMTD), F e n t i n A c e t a t e (FA), C h l o r a m p h e n i c o l (Chloram), C y c l o h e x i m i d e ( C y c l o ) o r PCNB Days a f t e r treatment

B i o c i d e added TMTD

FA

Chloram.

(% o f a p p l i e d 0 2 6 10 20

100 103 105 107 73

100 100 102 103 72

NP=non-history s o i l ,

y

Cyclo.

Z

PCNB

NP

100

100 96 90

P

y

remaining)

100 100 103 100 100

100

-

-

11

-

-

3 0

P = s o i l t h a t a c q u i r e d enhanced

100 100 5

9k 79

-

degradation.

Data adapted from Ref. 18 P e r s i s t e n c e o f Enhanced

Degradation

The p e r s i s t e n c e o f enhanced d e g r a d a t i o n c a p a c i t y i n the s o i l under l a b o r a t o r y c o n d i t i o n s and i n the f i e l d has been e x p l o r e d t o a c e r t a i n e x t e n t . I t has been shown t h a t enhanced d e g r a d a t i o n o f MBC l a s t e d i n the f i e l d f o r a t l e a s t 2 y e a r s and i n the l a b o r a t o r y f o r 3 y e a r s ( 1 1 ) . S i m i l a r r e s u l t s were o b t a i n e d w i t h diphenamid. S o i l t h a t was t r e a t e d w i t h diphenamid i n 1982 and a g a i n i n 1984, was found one



9.

AHARONSON ET AL.


123

y e a r l a t e r t o have d e v e l o p e d enhanced d e g r a d a t i o n under p r a c t i c a l f i e l d c o n d i t i o n s and p r e s e r v e d i t f o r a t l e a s t one y e a r . In t h e l a b o r a t o r y , some o f t h e d i p h e n a m i d - t r e a t e d s o i l s l o s t t h e i r a c t i v i t y a f t e r 1 o r 2 y e a r s , b u t t h e enhanced d e g r a d a t i o n c o u l d be r e s t o r e d i n these s o i l s much f a s t e r than i t took t o d e v e l o p enhanced d e g r a d a t i o n i n t h e f i r s t p l a c e i n t h e p r e v i o u s l y n o n - t r e a t e d s o i l . H a n d l i n g and s t o r a g e c o n d i t i o n s o f t h e s o i l i n t h e l a b o r a t o r y are c r i t i c a l f o r p r e s e r v i n g the a c t i v i t y . In mixed b a c t e r i a l c u l t u r e s t h e a c t i v i t y o f t h e diphenamid d e g r a d e r s was r a p i d l y l o s t . W i t h i n 2 weeks o f t h e f i r s t diphenamid treatment, the m i c r o b i a l degraders i n the c u l t u r e l o s t t h e i r c a p a b i l i t y t o degrade diphenamid. The a d d i t i o n o f v a r i o u s n u t r i e n t s , o r o f n o n t r e a t e d s o i l as a s o u r c e o f n u t r i e n t s , d i d n o t r e s t o r e d e g r a d a t i o n . W i t h MBC t h e r e s u l t s were somewhat d i f f e r e n t . The a c t i v i t y o f t h e d e g r a d e r s was m a i n t a i n e d f o r a l o n g e r p e r i o d o f time, b u t t h e r e were f l u c t u a t i o n s i n t h e a c t i v i t y , upon subsequent t r a n s f e r s , r a n g i n g from almost no d e g r a d a t i o n t o v e r y r a p i d degradation (19). F a i l u r e t o preserve the c a p a b i l i t y f o r degradation o f diphenamid i n t h e mixed b a c t e r i a l c u l t u r e might be one o f t h e r e a s o n s f o r t h e f a i l u r e t o i s o l a t e a pure c u l t u r e o f i n d i v i d u a l s t r a i n s o f b a c t e r i a c a p a b l e o f d e g r a d i n g diphenamid. S e v e r a l o t h e r r e c e n t s t u d i e s r e p o r t e d s i m i l a r o b s e r v a t i o n s , i n which t h e b a c t e r i a l c u l t u r e from s o i l w i t h enhanced d e g r a d a t i o n was v e r y a c t i v e b u t i n d i v i d u a l a c t i v e s t r a i n s c o u l d n o t be i s o l a t e d . Such r e p o r t s i n c l u d e d t h e h e r b i c i d e l i n u r o n (21), t h e f u n g i c i d e s i p r o d i o n e (22) and m e t a l a x y l ( 2 3 ) , and t h e i n s e c t i c i d e a l d i c a r b (2M). I t i s p o s s i b l e t h a t f a i l u r e t o i s o l a t e b a c t e r i a l d e g r a d e r s may be a s s o c i a t e d w i t h p e s t i c i d e s i n which t h e b a c t e r i a l d e g r a d e r s f a i l t o grow on t h e r e s p e c t i v e p e s t i c i d e as t h e s o l e c a r b o n s o u r c e . Pathways o f D e g r a d a t i o n and Enzymatic R e a c t i o n s A s s o c i a t e d w i t h Enhanced D e g r a d a t i o n o f Carbendazim and Diphenamid D a t a on t h e pathways and t h e enzymatic r e a c t i o n s i n v o l v e d i n enhanced d e g r a d a t i o n and t h e s p e c i f i c i t y o f t h e enzymes a r e i m p o r t a n t f o r b e t t e r u n d e r s t a n d i n g o f t h e development o f enhanced d e g r a d a t i o n . S t u d i e s w i t h MBC and diphenamid and w i t h some o f t h e i r d e g r a d a t i v e a n a l o g s have r e v e a l e d t h e f o l l o w i n g i n f o r m a t i o n ( T a b l e I I ) . MBC and i t s m e t a b o l i t e 2-AB were r a p i d l y degraded i n s o i l t h a t d e v e l o p e d enhanced d e g r a d a t i o n o f MBC. The d e g r a d a t i o n o f MBC and 2-AB i n t h e n o n - h i s t o r y s o i l , d u r i n g t h e time o f t h e experiment, as shown i n T a b l e I I was n e g l i g i b l e . On t h e o t h e r hand, t h e r a t e o f degradation o f the d e r i v a t i v e benzimidazole o r o f the f u n g i c i d e t h i a b e n d a z o l e was n o t enhanced i n t h e MBC-history s o i l . C r o s s enhancement was o b s e r v e d o n l y between MBC and 2-AB. These f i n d i n g s s u g g e s t e d t h a t t h e r e a c t i o n a s s o c i a t e d w i t h enhanced d e g r a d a t i o n o f MBC does n o t i n v o l v e r i n g c l e a v a g e , r i n g s u b s t i t u t i o n o r h y d r o x y l a t i o n . The r e a c t i o n seems t o i n v o l v e t h e amino moiety t h a t i s attached to the imidazole r i n g . The d a t a on diphenamid showed c r o s s enhancement o n l y between diphenamid and i t s monodemethylated d e g r a d a t i o n p r o d u c t ( d i p h e n M-l). The d e g r a d a t i o n o f t h e b i d e m e t h y l a t e d d e r i v a t i v e ( d i p h e n M-2) was n o t enhanced when d i p h e n M-2 was a p p l i e d t o t h e


124

ENHANCED BIODEGRADATION O F PESTICIDES IN T H E ENVIRONMENT

Table Π. Degradation of M B C , Diphenamid and their Derivatives in Soil and by Mixed Bacterial Cultures, Derived from Soils with Enhanced Degradation Capacity to Either Pesticide, Respectively

Degradation in mixed bacterial culture


Chemical name and structure

Days of incubation 0

4

8

( % of applied ) Diphenamid

3

CH-C-N^

CH

O ' N-Methyl-2,2-diphenyl acetamide(diphenM-l)

2,2-Diphenylacetamide (diphen M-2)

CD \

cH

\ " "

'

N

N

'

C

H

x

H

H

3

3

100

72

100

65

100

100

5

103

Degradation in soil Days of incubation 0

A

12

( % of applied ) Carbendazim (MBC)

2-Aminobenzimidazole

(QCJ^ " ^ 2

Benzimidazole

100

20

0

100

6

0

100

92

Data was adapted from Ref 18 and 19


9.

diphenamid-history II)

neither

derivative a

soil was

degradation, The

bidemethylated or

than

diphen M - l , i t s

culture

M-2 was

not

in

with

soil

enhanced

in

the

soil

have

demethylation an

oxidative

therefore soil

might

be

induction

of

oxidative as

the

resistance

by

Fungi

been

shown

by for

degradation, (26),

as

(21)

with

not

in

more

suggested

5).

the

slowly

that

degrades

diphen

diphenamid

(18). proceeded

mammals

function

of

of

is

mono-oxidases

by

or

the

It

is

soil the

by

the

enhanced

pesticide

usual

as

the

to

weeds,

consider

to

the

in

analogous

insects

One m i g h t

known

oxidases.

diphenamid i n

as

and

via

demethylation

considered

to

(Figure

detected

degraded

degradation

resistance

in

enhanced

concentration

diphenamid i n

mixed

pesticides

diphenamid by degradation

several

that

in

several

such

linuron

the

be

diphenamid

well

by

the

defined

pest.

of

soils

fungi

mammals,

analogues

of

instead

and w i t h

degradation

In by

processes.

rate

was

of

induction

buildup of

their

months

cultures

to

target

it

extract

degradation

could

probably degrade

but

several

with

of

soil

its

was

capacity

enhanced

population the

the

mediated

resistance

bacterial

the

metabolism

molecule.

associated

of

degradation

enzymes,

that

that

from

microorganism that

the

Such a mechanism

development

soil

the

process,

suggested

bacteria.

on

shown

of

from

monodemethylated

which

M-2)

(Table

in

diphenamid a p p l i e d

(diphen

degradation

reports

in

total

and s i n c e

absence the

The

conditions

degraded

product

obtained

culture,

extracts,

p r o d u c e d by

2 weeks.

was

the

derivative

soil

were

a degradation

that of

experimental

derivatives

first

as

Fusarium 3k%

these

its

the

culture

6 days

Previous


during

and i n

after in

Under

identified

mixed b a c t e r i a l

reached

soil.

diphenamid nor

non-history

125


AHARONSON E T AL.

in

days,

developed

carbendazim

soil

(19),

oxidative is

observed

enhanced in

much

with

slower:

bacterial

degradation.

bacterial

pesticides

and m e t a z a c h l o r

same

the

as

comparison with other

the

soils

Slower

degradation with

isofenphos

has

enhanced

(25),

lindane

(16).

Conclusions Enhanced

degradation

effectiveness consequences is

often

of

of

to

pesticides

additional

abandoning crop

the

by

in

the of

use

soil,

of

of

rapid the

rotation.

repeated

buildup,

and

example

In

the

loss

of

their

undesirable such

cases

monoculture

same p e s t i c i d e s .

populations

of

This

soilborne

is

pests

pathogens. A shift

enhanced to

an

accompanied

analogous and

is

be

in

microbial

degradation

associated

Moreover,

the is

reaction

soil

in

development several

of

Because

of

analogy

for

of

which

regarding the

the from

that

might

pests. the

rather shown

than

that

induction of be

acquired

MBC a n d E P T C

oxidative

to

Such a p o s s i b i l i t y

persistence

of

such

seems

fungi.

enhanced

an

analogous

with

the raises

changes

and

the

process.

many u n c e r t a i n t i e s the

and pathogens,

and

Therefore,

approach

the

linked with in

soils

degraders,

diphenamid have

bacteria,

resistance

in

toward diphenamid,

bacterial

apparently

questions

generalization

simple

with

results

degradation

degradation

capacity

their

buildup of control, for

about

this

populations should

dealing

with

always

of be

enhanced

phenomenon, soilborne kept

in

the

pests mind.

degradation

should



126

E N H A N C E D BIODEGRADATION O F PESTICIDES IN T H E E N V I R O N M E N T

50

2

0

Figure

5.

Disappearance

demethylated Fusarium

metabolite

4 6 DAYS OF INCUBATION o f diphenamid (desmethyl

8

and the formation

diphenamid,

diphen

of

its

M-l) i n

culture.


9. be

similar

to

detect

means,

and

developed

to the

that

taken

problem

only

as

enhanced

127


AHARONSONETAL.

the

for

well last

controlling

in

advance,

step

to

to

treat

soilborne develop the

soil

pathogens,

i.e.,

preventative which

has

degradation.


Literature Cited 1. Roeth, F.W. Rev. Weed Res. 1986, 2, 45-65. 2. Kaufman, D. D.; Katan, J.; Edwards, D.F.; Jordan, E . J . in: Agricultural Chemicals of the Future, Hilton, J.L. Ed.; Rowman & Alanheld: New Jersey, 1985, pp. 437-451. 3. Katan, J.; Aharonson, N. in: Ecological Studies; Vol 73, Toxic Organic Chemicals in Porous Media, Gerstel, Ζ.; Chen, Y . ; Mingelgrin, U.; Yaron, B. Eds. Springer Verlag, 1989,pp.193-207. 4. Felsot, A.S. Ann. Rev. Entomol. 1989, 34, 453-476. 5. Suett, D.L; Walker, A. Aspects of Appl. Biol. 1988, 17, 213-222. 6. Tollefson, J. Proc. Brit. Crop Prot. Conf.-Pests & Diseases. 1986, 3, p. 1131-1136. 7. Read, D.C. Agric. Ecosystems Environ. 1986, 15, 51-61. 8. Obrigawitch, T . ; Martin, A.R.; Roeth, F.W. Weed Sci. 1983, 31, 187-192. 9. Avidov, E . ; Aharonson, N.; Katan, J. Weed Sci. 1988, 36, 519-523. 10. Racke, K.D.; Coats, J.R. J. Agric. Food Chem. 1988, 36, 193-199. 11. Yarden, O.; Aharonson, N.; Katan, J. Soil Biol. Biochem. 1987, 19, 735-739. 12. Walker, Α.; Brown, P.A.; Entwistle, A.R. Pestic. Sci. 1986, 17, 183-193. 13. Tal, Α.; Rubin, B.; Katan, J.; Aharonson, N. J. Weed S c i . , 1989, 37, 434-439. 14. Avidov, E . ; Aharonson, N.; Katan, J.; Rubin, B.; Yarden, O. Weed Sci. 1985, 33, 457-461. 15. Ingham, E.R. Crop Protection. 1985, 4, 3-32. 16. Allen, R.; Walker, A. Pestic. Sci. 1988, 22, 297-305. 17. Farley, J.D.; Lockwood, J.L. Phytopathology, 1968, 58, 714-715. 18. Avidov, E . ; Aharonson, N.; Katan, J. Weed Sci. 1990, 38, (in press). 19. Yarden, O.; Salomon, R.; Katan, J.; Aharonson, N. Can. J. Microbiol. 36, 1990 (in press). 20. Tal, A. Ph.D Tesis, The Hebrew University of Jerusalem, Israel 1988. 21. Glad, G.; Goransson, B.; Popoff, T . ; Theander, O.; Torstensson, N.T.L. Swedish J. Agric. Res. 1981, 11, 127-134. 22. Head, I.M.; Cain, R.B.; Suett, D.L.; Walker, A. Brighton Crop Protection Conf.-Pests & Diseases. 1988, p. 699-704. 23. Ana Maria Bailey; Coffey, M.D. Can. J. Microbiol. 1986, 32, 562-569. 24. Read, D.C. J. Econ. Entomol. 1987, 80, 156-163. 25. Racke, K.D.; Coats, J.R. J. Agric. Food Chem. 1987, 35, 94-99. 26. Wada, H . ; Senoo, K.; Takai, Y. Soil Sci. Plant Nutr. 1989, 35, 71-77. RECEIVED January 22, 1990


Enhanced Biodegradation of Pesticides in the Environment - American

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